Method and composition for hyperthermally treating cells

ABSTRACT

A method for the hyperthermia treatment of tissue in a target site applies a heat source to kill cells without protein denaturization. The method introduces an encapsulated dye that is released at a selected temperature in the target site to indicate that a threshold temperature has been reached to hyperthermally treat the tissue. In one embodiment, the composition releases the dye at a temperature of 42° C. to 50° C., and preferably about 45° C. to 49° C. The composition which can be a liposome composition encapsulating the dye can be introduced to the bloodstream of the patient to flow through the target site.

This application is a divisional application of Ser. No. 10/073,863,filed Feb. 14, 2002 now U.S. Pat. No. 7,101,571.

FIELD OF THE INVENTION

The present invention relates to a method and composition forhyperthermally treating cells at a site in the body. More particularly,the present invention relates to a method for treating cells at a targetsite in the body, such as at a lens capsule of an eye, tumors, andexudative ARM (age related macular degeneration) by applying thermalenergy to the target site to heat the cells to a temperature which willkill the cells or impede cell multiplication without exceeding theprotein denaturation temperature of the tissue.

BACKGROUND OF THE INVENTION

Several techniques currently exist for treating cells at a selected sitein the body with heat or chemicals to kill or impede multiplication ofthose cells to prevent undesired cell proliferation. For example,numerous types of chemotherapy drugs exists which, when injected into atumor or delivered systemically to a patient, attack and kill cancerouscells to prevent them from further multiplying.

Radiation techniques can also be used to kill cancerous or otherundesired cells. Cell death begins to occur when the cells are heated toa temperature of about 5° C. or more above the normal body temperatureof 37° C. Applying radiation to a localized site in the body, such as atumor or other area containing undesired cells, can heat the cells atthe site to temperatures in excess of 60° C. Such high temperaturescauses a phenomenon known as protein denaturation to occur in the cells,which results in immediate cell death. Accordingly, radiation therapyhas been suitable in successfully treating certain types of cancers andother diseases involving uncontrolled cell growth.

Other types of heating techniques, such as the use of probes orcatheters to provide localized heat to a site of interest also exist.Like radiation therapy, these techniques also heat the cells to atemperature sufficient to cause protein denaturation in the cells tothus kill the cells quickly.

Photosensitive chemicals are also used to kill cells at certain sites ofinterest in the body. For example, a photosensitive chemical can beinjected directly into a site of interest to expose cells at that siteto the chemical. A light emitting source, which emits light at awavelength that will activate the photosensitive chemical, is thenfocused on the site of interest. Accordingly, the light activates thephotosensitive chemical that has been absorbed by or is otherwisepresent in the cells of interest. The activated chemical kills thecells, which thus prevents undesired cell proliferation.

Although the techniques mentioned above can be suitable for preventingcertain types of cell proliferation and certain sites in the body,several drawbacks with these techniques exist. For example, often theuse of chemotherapy drugs alone to treat a tumor or cancerous site isinsufficient to kill the undesired cells. Moreover, the chemotherapydrugs also kill many normal healthy cells along with the cancerouscells, which can adversely affect the patient's health.

The use of radiation in conjunction with chemotherapy can have a moredetrimental effect on the cancerous cells. However, as withchemotherapy, radiation often kills normal healthy cells, such as thosein front of or behind the site of interest, along with the cancerouscells. Moreover, the intense heating of the cells can cause the cells tocoagulate and thus block the capillaries at the site of interest. Theblocked capillaries therefore prevent chemotherapy drugs from reachingthe site of interest.

One example of a method of chemically treating a target site isdisclosed in U.S. Pat. No. 6,248,727 to Zeimer. This method delivers aliposome containing a fluorescent dye and tissue-reactive agent. Theliposome is administered intravenously to flow to the locus in the eyeof the patient and the site is non-invasively heated to release the dyeand the tissue-reactive agent. The dye is fluoresced to observe thepattern of the fluorescence. The tissue-reactive agent is activated tochemically damage and occlude the blood vessel. The liposomes areselected to release the dye at a temperature of 41° C. or less withoutcausing thermal damage to the blood vessel.

In addition, the above techniques have not been used to prevent unwantedcell proliferation at certain locations in the eye, such as at theretina or at the lens capsule. Because the retina is very sensitive,conventional radiation techniques can be too severe to treat cancerouscells on, in or under the retina.

Also, after cataract surgery, a phenomenon known as capsularopacification and, in particular, posterior capsular opacification canoccur in which the epithelial cells on the lens capsule of the eyeexperience proliferated growth. This growth can result in the cellscovering all or a substantial portion of the front and rear surfaces ofthe lens capsule, which can cause the lens capsule to become cloudy andthus adversely affect the patient's vision. These cells can be removedby known techniques, such as by scraping away the epithelial cells.However, it is often difficult to remove all of the unwanted cells.Hence, after time, the unwanted cells typically will grow back, thusrequiring further surgery.

Accordingly, a need exists for a method for hyperthermally treatingtissue and preventing unwanted cell proliferation at sites in the body,especially at sites in the eye such as the retina, choroid and lenscapsule, which does not suffer from the drawbacks associated with theknown techniques discussed above.

SUMMARY OF THE INVENTION

The present invention is directed to a method of hyperthermally treatingtissue by heating the tissue above a temperature which kills cells inthe tissue. In particular, the invention is directed to a method ofheating tissue above a temperature effective to treat the tissue withoutdenaturing the protein.

Accordingly, a primary aspect of the invention is to provide a methodfor heating tissue at least to a temperature sufficient tohyperthermally treat the tissue.

Another aspect of the invention is to provide a method of hyperthermallytreating tissue to a temperature sufficient to kill cells in the tissueand at a temperature below the protein denaturization temperature of thetissue.

A further aspect of the invention is to provide a method ofhyperthermally treating tissue, where the tissue includes or is providedwith a temperature indicator to indicate a hyperthermally effectivetemperature of the tissue.

Still another aspect of the invention is to provide a method ofhyperthermally treating tissue where a temperature indicator compositionis introduced into the tissue or bloodstream near the tissue to indicatea tissue temperature effective to hyperthermally treat the tissue and atemperature indicator to indicate a tissue temperature above a proteindenaturization temperature of said tissue.

A further aspect of the invention is to provide a method ofhyperthermally treating tissue by introducing a temperature indicatorinto the tissue and heating the tissue to a temperature where thetemperature indicator can be detected. In a preferred embodiment, thetemperature at which the indicator can be detected is a temperatureeffective to hyperthermally treat the tissue and is at a temperaturebelow the protein denaturization temperature.

A further aspect of the invention is to provide a method of heating anddetecting a temperature of a tissue between a first temperature and asecond temperature. The method introduces a temperature indicator intothe tissue. The temperature indicator includes a first dye that can bedetected at the first temperature to indicate that the first temperaturehas been reached, and a second dye that can be detected at the secondtemperature to indicate that the second temperature has been reached.

Still another aspect of the invention is to provide a temperatureindicating composition for introducing into a tissue to be thermallytreated. The composition includes a first dye encapsulated in a heatsensitive liposome where the first dye is releasable at a temperatureeffective to hyperthermally treat the tissue and at a temperature belowthe protein denaturization temperature. The composition also includes asecond dye encapsulated in a second liposome where the second dye isreleasable at a temperature at or above the protein denaturizationtemperature.

Another aspect of the invention is to provide a method to hyperthermallytreat tissue to kill the tissue cells substantially without proteindenaturization of the tissue where the tissue includes a heat sensitiveliposome containing a temperature indicating dye and a temperatureactivated bioactive compound. The tissue is heated to release the dyefrom the liposome to indicate a thermally effective temperature to killcells in the tissue at a temperature below the protein denaturizationtemperature. The heat applied to the tissue simultaneously releases thebioactive compound to treat the tissue.

The various aspects of the invention are basically attained by providinga method of hyperthermally treating tissue in an animal. The methodcomprises the step of introducing a temperature indicating substanceinto the bloodstream of the animal to flow through a target site. Thetemperature indicating substance includes a fluorescent dye encapsulatedwithin a heat sensitive liposome. The fluorescent dye is releasable fromthe liposome at a temperature of at least 41° C. A heat source isapplied to the target site and the target is hyperthermally heated to atleast 41° C. to release and fluoresce the dye and to hyperthermallytreat the target site for a time sufficient to kill cells in the tissue.

The aspects of the invention are also attained by providing a method ofdetecting a threshold temperature and of hyperthermally treating tissuein an animal. The method comprises the step of introducing a firstfluorescent dye encapsulated in a first heat sensitive liposome into thebloodstream of an animal in a location to flow through a target site inthe animal. The first fluorescent dye is releasable from the first heatsensitive liposome at a temperature of at least 41° C. The target siteis heated to a temperature to release the first fluorescent dye and thefirst fluorescent dye is fluoresced to indicate and visualize a tissuetemperature of at least 41° C. Heating of the target site is continuedat a temperature of at least 41° C. for a time sufficient tohyperthermally treat the tissue.

The aspects of the invention are further attained by providing a methodof hyperthermally treating tissue of an animal. The method comprises thestep of introducing a temperature indicating substance into thebloodstream of the animal to flow through a target site. The temperatureindicating substance includes a first fluorescent dye encapsulated in afirst temperature sensitive liposome. The first fluorescent dye isreleasable from the first liposome by heating to a temperature of atleast 42° C. A second fluorescent dye encapsulated in a secondtemperature sensitive liposome is also included. The second fluorescentdye is releasable from the second liposome by heating to a temperatureof at least 50° C. The target site is heated to a temperature of atleast 42° C. The first fluorescent dye is fluoresced to indicate aneffective temperature for hyperthermally treating the tissue withoutreleasing the second fluorescent dye from the second liposomes.

These and other aspects of the invention will become apparent to oneskilled in the art in view of the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawing, in which:

The FIGURE is a schematic diagram of one embodiment of the inventionshowing a probe for hyperthermally treating tissue and visualizing a dyein the target site.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method and composition forhyperthermally treating tissue. In particular, the invention is directedto as method for heating tissue above a temperature effective to killtissue cells or inhibit multiplication of cells below the proteindenaturization temperature of the tissue.

The method of the invention introduces a composition into thebloodstream of the body in a location to flow into or through a targetsite to be treated. A heat source is applied to the target site to heatthe tissue in the target site for a time sufficient to hyperthermallytreat the tissue and activate the composition. As used herein, the term“hyperthermal” refers to a temperature of the cell or tissue that killsor damages the cells without protein denaturization.

The composition contains a temperature indicator that is able to providea visual indication when a minimum or threshold temperature is attainedthat is sufficient to hyperthermally treat the tissue. It is a featureof the invention to provide a method of heating tissue in a target siteto a hyperthermally effective temperature and to provide a visualindication that a temperature of at least 41° C., and preferably atleast 42° C. is attained. In one embodiment, the composition includes asecond temperature indicator to provide a visual indication when aprotein denaturization temperature is attained thereby providing anindication that a maximum desired temperature is exceeded. The heatsource can be applied to the tissue so that the composition provides anindication that a thermally effective temperature is attained that isbelow the protein denaturization temperature of the tissue.

In one embodiment of the invention, the method introduces a compositionto a target site, where the composition includes a fluorescent dye thatis encapsulated in a heat sensitive particle, such as a liposome. Thedye is a fluorescent dye that can be excited to fluoresce and beobserved or visualized by the operator. Preferably, the heat sensitiveliposomes are formed to rupture or release the fluorescent dye at atemperature at least equal to the temperature necessary to kill cells inthe tissue and at a temperature below the protein denaturizationtemperature. The composition containing the heat sensitive liposomesencapsulating the fluorescent dye is introduced into the bloodstream toflow to or through the target site. The amount of the liposomecomposition is introduced in an amount effective to be released in ornear the target site and to excited and visualized by the exciting lightsource and the visualizing device. The composition containing the dyecan be injected in a single dose into the bloodstream or injectedcontinuously to supply a continuous flow of the composition through thetarget site. The amount of the composition introduced can vary dependingon the target site and the length of time that the dye is to be excited.A light or energy source is continuously applied to the target site toexcite the dye and to cause the dye to fluoresce when released from theliposomes. An imaging device is used to capture the fluorescing lightfrom the dye to provide a visual indication that the dye is released.The release temperature of the liposomes are selected to release the dyeat a predetermined temperature so that when the dye is fluoresced andvisualized, the visualization provides the operator with an indicationthat the release temperature in the target site has been attained. Inone embodiment, the liposome composition is injected into the bloodstream so that the composition is able to provide a continuous supply ofthe dye for fluorescing during the hyperthermal treatment. In thismanner the operator is provided with a continuous indication that asufficient temperature is being maintained.

The method of the invention is primarily directed to a method of heatingtissue and cells in the tissue of an animal, particularly a humanpatient, at least to the temperature sufficient to kill or damage thecells. Cell death or cell damage is known to occur when the tissue cellsare heated to a temperature of about 5° C. above the normal bodytemperature of 37° C. Therefore, the method of the invention heats thecells in the tissue to a temperature of about 41° C., and preferably atleast 42° C. for a time sufficient to kill or damage the cells.Preferably, the heat source is applied to minimize unnecessary damage tothe surrounding cells and tissue.

In one embodiment of the invention, the tissue is heated to atemperature of at least 41° C. and preferably in the range of at leastabout 42° C. to about 50° C. Heating the tissue to at least 42° C.ensures that a sufficient temperature is obtained to thermally treat thetissue and the cells effectively. Preferably, the tissue is heated to atemperature below the protein denaturization temperature of the tissue.Protein denaturization begins to occur at about 50° C. to 51° C. andoccurs rapidly at temperatures of about 60° C. Preferably, the tissue isheated to a temperature of less than 60° C. and more preferably to atemperature of about 50° C. or less.

In one preferred embodiment, the tissue and the cells are heated to atemperature of about 47° C. to about 49° C. for a time sufficient tokill or damage the cells without protein denaturization. The length oftime that the tissue is heated will depend on the location of the targetsite, the size and dimensions of the target site, the desired depth ofpenetration of the heat and the desired extent of thermal treatment ordamage of the tissue and cells in the target site. Typically, the heatsource is applied for several minutes. In one embodiment, the heatsource is applied for about 1 to 15 minutes, and typically about 5 to 10minutes.

The heat source can be applied to a variety of the areas in the bodywhere the hyperthermal treatment is desired. The target site can betumors, organs, muscles and soft tissue. Examples of a target siteinclude blood vessels and arteries, esophagus and eyes. In oneembodiment, the method is suitable for hyperthermally treating theepithelial cells on the lens of the eye after cataract surgery. Othertarget sites include the retina and the choroid.

The target site is heated to the desired temperature to hyperthermallytreat the target site using standard heating instruments and equipmentfor heating tissue and standard equipment for visualizing the dye in thetarget site that has been released from the heat sensitive particles.For example, the heating equipment preferably includes suitable heat orenergy source that is able to focus the heat or energy on the target andis able to control heat and temperature of the tissue. The heat sourcecan be an electrical resistance heating element, or an indirectly heatedelement. The heating device can also have an energy source for producingheat at the target site, such as a radio frequency (RF) device,ultrasonic generators, laser, or infrared device. One example of an RFgenerator device for hyperthermally treating tissue in a selected targetsite is disclosed in U.S. Pat. No. 6,197,022, which is herebyincorporated by reference in its entirety. Examples of suitableultrasonic devices for delivering ultrasonic hyperthermia are disclosedin U.S. Pat. Nos. 4,620,546, 4,658,828 and 4,586,512, the disclosures ofwhich are hereby incorporated by reference in their entirety.

In one embodiment, the heat source includes a probe having a tip withthe heating element or energy emitting element attached thereto. Theenergy emitting element can be an optical fiber operatively connected toa laser, infrared or ultraviolet light source. The probe preferablyincludes a suitable control mechanism for manipulating the probe to thetarget site and a control for controlling the energy applied to thetarget site.

A suitable device for hyperthermally treating the tissue in a targetsite is shown in the figure. The device 10 includes a probe 12 having anoptical fiber 14 with a distal end 16 for emitting a laser light to heatthe tissue 17. Preferably, the end 16 of optical fiber 14 can focus thelight source on the target site 17. Optical fiber 14 is connected tolaser generator 18 that is able to generate a laser beam of sufficientintensity and within wavelength for hyperthermally treating tissue to atemperature of at least 41° C. and preferably at least 42° C.

In a preferred embodiment, probe 12 includes a second optical fiber 20having a distal end 22 and a third optical fiber 24 having a distal end26. Optical fiber 20 is operatively connected to a light source 28, suchas a laser, that is able to emit a light beam having a wavelengthcapable of fluorescing a fluorescent dye in the target area when the dyeis released from the heat sensitive particles. Optical fiber 24 isoperatively connected to a suitable imaging device 30 for capturing thefluoresced light from the excited dye and visualizing and producing animage of the fluorescing dye in the target site. Imaging device 30 canbe a CCD or a device equivalent to a funduscope. An example of asuitable funduscope is disclosed in U.S. Pat. No. 4,891,043 to Zeimer,which is hereby incorporated by reference in its entirety.

In another embodiment of the invention, the probe can include a heatingelement or a device for receiving a heated fluid that can transfer theheat to the tissue in the target site. The probe can include anexpandable bladder member for receiving a heated fluid delivered from afluid-heating source. In still another embodiment, the expandablebladder includes a permeable portion so that the heated fluid can beapplied directly to the target site. A suitable aspirating device ispreferably included to remove the excess heating fluid when applieddirectly to the target site.

In one embodiment, the target site is the retina or choroid in the eyeof the patient. The heating and visualizing instrument includes a lasercapable of focusing a laser beam on the target site where the laser beamhas a wavelength and intensity to heat the cells to a temperature of atleast 42° C. In one embodiment, the laser heats the cells to atemperature of 50° C. or below and preferably to about 42° C. to 50° C.The instrument also includes or is used in combination with a funduscopeto excite or fluoresce the dye that has been released in the target siteto capture and visualize the fluorescing dye. A funduscope that can beused is disclosed in U.S. Pat. No. 6,248,727, which is herebyincorporated by reference in its entirety. The laser source is selectedto provide sufficient energy to heat the tissue in the target site tothe desired temperature.

The fluorescent dye is encapsulated in a suitable heat sensitiveparticle and introduced into the patient in a location to be visualizedin the target site. The heat sensitive particles can be microcapsules,or nanocapsules that are able to release the dye at a temperature ofabout 41° C., and preferably 42° C. or higher. In preferred embodiments,the fluorescent dyes are incorporated into heat sensitive liposomes thathave a phase transition temperature at the temperature of hyperthermia.In one embodiment, the liposomes have a phase transition temperaturewithin the desired temperature range that tissue or cells are to beheated.

In one embodiment, the liposomes have a phase transition temperature ofat least 41° C. and preferably at least 42° C. In a preferredembodiment, the liposomes have a phase transition temperature of about45° C. to about 50° C.

The liposomes can be made by various processes as known in the art. Thephase transition temperature of the phospholipid is selected to controlthe temperature that the dye and other components are released from theliposomes. Phospholipids are known to have different phase transitiontemperatures and can be used to produce liposomes having releasetemperatures corresponding to the phase transiture of the phospholipids.Suitable phospholipids include, for example, dimyristoylphosphatidylcholine having a phase transition temperature of 23.9° C.,palmitoylmyristoylphosphatidyl choline having a phase transitiontemperature of 27.2° C., myristolypalmitoylphosphatidyl choline having aphase transition temperature of 35.3° C., dipalmitoylphosphatidylcholine having a phase transition temperature of 41.4° C.stearoylpalmitoylphosphatidyl choline having a phase transitiontemperature of 44.0° C., palmitoylstearolyphosphatidyl choline having aphase transition of 47.4° C., and distearolyphosphatidyl choline havinga phase transition temperature of 54.9° C. Another suitable phospholipidis a synthetic C₁₇ phosphatidyl choline from Aventi Inc. having a phasetransition temperature of about 48° C.-49° C.

The phase transition temperature and the release temperature of theliposomes can be selected by combining the different phospholipidsduring the production of the liposomes according to the respective phasetransition temperature. The phase transition of the resulting liposomemembrane is generally proportional to the ratio by weight of theindividual phospholipids. Thus, the composition of the phospholipids areselected based on the respective phase transition temperature so thatthe phase transition temperature of the liposome membrane will fallwithin the selected range. By adjusting the phase transition temperatureof the liposome membrane to the selected range, the temperature at whichthe liposomes release the dyes and other components can be controlledduring hyperthermia.

The liposomes in one embodiment of the invention are preferably preparedso that the liposome membrane has a phase transition temperature of atleast 42° C., and preferably about 42° C. to about 50° C. In a preferredembodiment, the liposomes leak or rupture at a temperature of about 49°C. or less, and typically between about 45° C. and 49° C. In oneembodiment, the phospholipids have saturated acyl groups. For example,glycerophospholipids can be used that have two acyl groups having 8 ormore carbon atoms and where at least one of the acyl groups have atleast 10 carbon atoms and typically 12-18 carbon atoms. Examples ofsuitable phospholipids include hydrogenated lecithin from plants andanimals, such as egg yolk lecithin and soybean lecithin. Thephospholipid can also be phosphatidyl choline produced from partial orcomplete synthesis containing mixed acyl groups of lauryl, myristoyl,palmitoyl and stearoyl.

The liposomes can be prepared from a mixture of dipalmitoylphosphatidylcholine and disteroylphosphatidyl choline in a weight ratio of 95:5 toabout 5:95 and generally about 80:20 to about 20:80. In one embodiment,the liposomes are made from a mixture of dipalmitoylphosphatidyl cholineand disteroylphosphatidyl choline in a ratio of 45:55 to about 55:45provide a phase transition temperature of about 46° C. to about 49° C.

The liposomes of the invention can be prepared by standard processes asknown in the art. The liposomes can be unilamellar or multilamellar andhave a particle suitable for delivering the dye to the target site. Inone embodiment, the liposomes have a particle size of a sufficientlysmall size to be introduced into the bloodstream of the patient in alocation near the target site to flow through the target site.

The liposomes can contain a suitable osmotic pressure controlling agentthat is physiologically acceptable to the patient. Examples includesodium chloride, sugars such as glucose, mannitol and sorbitol, andamino acids such as glycine, aspartic acid and glutamic acid. Examplesof suitable process for preparing liposomes are disclosed in U.S. Pat.No. 4,235,871 to Papahadjopoulos et al. and U.S. Pat. No. 4,522,803 toLenk, which are hereby incorporated by reference in their entirety.

The liposomes of the invention contain a dye that is able to fluoresceand that can be visualized in the target site by exciting with a lightsource that is amenable to the target site and the patient. Thefluorescent dye can be any fluorescent that is suitable forencapsulation and is physiologically compatible. Preferably, thefluorescent dye is quenched when encapsulated at an appropriateconcentration. The quenching concentration is a sufficiently highconcentration to mask or minimize detection of fluorescence whenilluminated by a fluorescing light source. The quenching concentrationcan be determined by routine experimentation as known in the art. Whenheated, the heat sensitive liposomes rupture or leak the dye and othercomponents of the liposome so that the dye is diluted in the target siteto a suitable concentration where the dye can be fluoresced andvisualized upon excitation by a suitable light source. Examples ofsuitable dyes include 6-carboxyfluorescein and its derivatives. Suitablefluorescent dyes can be excited by an emit light at wavelengths that arenot strongly absorbed by the tissue. Other suitable dyes includeindocyanin green and aluminum phthalocyanine tetrasulfonate. It will beunderstood that the fluorescing light source and the visualizinginstrument are selected according to the wavelength of the fluorescingdye to visualize the dye.

In one embodiment, the dye is selected to fluoresce in the presence of alight from an argon laser, a helium-neon laser or infrared laser.Preferably the dye is selected to be compatible with the exciting lightor laser source to fluoresce when subjected to the light or laser beam.A suitable dye is sold under the tradename D-275 by Molecular Probe,Inc. and fluoresces green when exposed to light from an argon laser at484 nm. A dye sold under the tradename D-1121 fluoresces orange whenexposed to a long wavelength laser light at 560-574 nm. On preferred dyeis an infrared excitable dye DiIc₁₈(7), which fluoresces at a wavelengthof 740-780 nm.

In one embodiment of the invention, a fluorescent dye is encapsulated inliposomes having a phase transition temperature of 42° C. to 50° C., andpreferably about 45° C. to 49° C. In another embodiment, the liposomeshave a phase transition temperature to release the dye at a temperatureof about 46° C. to about 49° C. The liposomes are injected into thebloodstream of the patient in a location where the liposomes flow to thetarget site. In some embodiments, the liposomes can be introduceddirectly to the target site intravenously, subcutaneously or topically.A hyperthermal heat source and a dye exciting light source are appliedto the target site. The hyperthermal heat source, which is preferably alaser light beam, is focused on the target site to heat the tissue andthe cells to a temperature of at least 42° C. to hyperthermally treatthe tissue and kill the cells. The hyperthermal heat source also heatsthe liposomes to a temperature at least equal to the phase transitiontemperature to release the dye. The fluorescing light source excites thedye so that the fluorescing dye in the target site can be detected andvisualized. By encapsulating the fluorescent dye in liposomes having aphase transition of at least 42° C., the detection of the fluorescingdye provides a positive indication to the operator that the desiredtissue temperature has been obtained that is necessary to hyperthermallytreat the tissue. The phase transition temperature of the liposomes isselected according to desired minimum temperature that the tissue is tobe heated. The hyperthermia energy source is applied to the target sitefor a time sufficient to treat the tissue to the desired level.Generally, the tissue is heated to a temperature of at least 42° C. for1-15 minutes and preferably 1-10 minutes.

In one preferred embodiment of the invention, the liposomes contain asuitable drug or photosensitizing agent. The drugs preferably show asynergistic effect when combined with the hyperthermia treatment of theinvention. The release of the drugs from the liposomes provide animproved targeting effect by releasing the drugs by the heat source inthe target site. Suitable drugs include antitumor agents such ascisplatin, carboplatin, tetraplatin and iproplatin. Suitable anticancerdrugs include adriamycin, mitomycin C, actinomycin, ansamitocin and itsderivatives, bleomycin, Ara-C, daunomycin, metabolic antagonists such as5-FU, methotrexate, isobutyl5-fluoro-6-E-furfurylideneamino-xy-1,2,3,4,5,6hexahydro-2,4-dioxopyrimidine-5-carboxylate. Other antitumor agentsinclude melpharan, mitoxantrone and lymphokines. The amount of theparticular drug entrapped in the liposomes are selected according to thedesired therapeutic dose and the unit dose.

Examples of suitable photosensitive (photosensitizer) agents includeaminolevolunic acid, porphyrine derivatives, porpurine derivatives,NPE-6, ATX-10, plant derived photosensitizers. Other syntheticsensitizers such as SNET₂ and Lutex can be used. Preferably, thephotosensitizers are used in non-toxic amounts. In other embodiments,the liposome compositions can contain liposomes that encapsulate ahyperthermic potentiating agent such as perfluorooctyliodide,perfluorotributylamine, perfluorotripropylamine, andperfluorooctylbromide. Examples of liposome encapsulated potentiatorsare disclosed in U.S. Pat. No. 5,149,319 to Unger, which is herebyincorporated by reference in its entirety. Other bioactive agents thatcan be delivered to the target site by encapsulating in liposomesinclude anti-inflammatory agents, antibiotics, antibacterial agents,antifungal agents, anti-neoplastic agents and antiparasitic agents.Examples of anti-neoplastic agents include aclacinomycins, chromycinsand olivomycins.

In another embodiment of the invention, the liposome compositioncontains a mixture of liposomes having different phase transitiontemperature to release the components at different temperatures. In oneembodiment, the liposome composition contains liposomes encapsulating afirst dye and having a phase transition temperature of 42° C. to about45° C. and liposomes encapsulating a second dye and having a phasetransition temperature of about 50° C. or higher. In one embodiment, thesecond dye is encapsulated in liposome that release the dye at atemperature range of 50° C. to 60° C. Preferably, the second dye is ableto fluoresce at different color than the first dye so that the dyes aredistinguishable. In this embodiment, the liposome composition isdelivered to the target and the target site is subjected to hyperthermiatemperatures. As the tissue in the target site is heated to at least 42°C., the first liposomes rupture or release the first dye so that thefirst dye is visualized and detected in the target site. The detectionof the first dye in the target site enables the operator to monitor thetemperature of the tissue in the target site and to indicate that ahyperthermal temperature has been attained in the tissue at the targetsite. During hyperthermia, it is difficult to determine and monitor theactual temperature of the tissue and care must be taken to avoidoverheating of the tissue and denaturization of the proteins. Inpreferred embodiments of the invention, the hyperthermal treatment doesnot exceed the protein denaturization temperature. In this embodiment,the second liposomes are selected to rupture or release the second dyeat or slightly below the protein denaturization temperature. In thismanner, the second dye is released and visualized to provide theoperator with an indication that the tissue is heated to the proteindenaturization temperature. The heat source is then adjusted by theoperator to reduce the energy applied to the target site to avoidprotein denaturization.

In another embodiment, the liposome composition can contain severalliposomes that can leak or rupture at different temperatures to releasethe dyes at incremental temperatures as the temperature of the targetsite increases. In one embodiment, the liposomes can be selected to leakor rupture the dye at 2° C. intervals between about 42° C. and 50° C.The dyes for each liposome can be different to fluoresce a differentcolor so that the different colors indicate a different temperature ofthe target site.

In other embodiments of the invention, the tissue in the target site canbe irradiated by beta radiation from strontium or iridium isotopes.Gamma radiation from p³², iodine-95, and palladium-90 can also be used.The radioactive isotopes can be delivered as small particles to thetarget site in combination with the hyperthermia treatment.

Although several embodiments have been chosen to illustrate theinvention, those skilled in the art will readily appreciate that variouschanges and modifications can be made without departing from the scopeof the invention.

1. A method of hyperthermally treating tissue in an animal, said methodcomprising the steps of: introducing a temperature indicating substanceinto the bloodstream of said animal to flow through said tissue in atarget site, said temperature indicating substance including afluorescent dye encapsulated within a heat sensitive liposome, saidfluorescent dye being releasable from said liposome at a temperature ofabout 45° C. to about 49° C., and applying a heat source to said tissueat said target site and hyperthermally heating said tissue in saidtarget site to at least 47° C. to release said dye and to hyperthermallytreat said tissue in said target site for a time sufficient to killcells in said tissue substantially without denaturing proteins in saidtissue, and fluorescing and visualizing said dye to indicate that apredetermined tissue temperature has been attained at said target site.2. The method of claim 1, wherein said liposome encapsulates a bioactivecompound, and said method comprises heating said liposome to releasesaid bioactive compound.
 3. The method of claim 2, wherein saidbioactive compound is heat activated.
 4. The method of claim 2, whereinsaid bioactive compound is an antiproliferative agent or an antitumoragent.
 5. The method of claim 2, wherein said bioactive compound isselected from the group consisting of cisplatin, carboplatin,tetraplatin, iproplatin, adriamycin, mitomycin C, actinomycin,ansamitocin and bleomycin.
 6. The method of claim 1, wherein said heatsource is a laser source, a microwave source, an infrared source, or anultrasonic source.
 7. The method of claim 1, wherein said heat source isa heated fluid source, and where said method comprises applying saidheated fluid to said target site.
 8. A method of detecting a thresholdtemperature and hyperthermally treating tissue in an animal, said methodcomprising the steps of: introducing a first fluorescent dyeencapsulated in a first heat sensitive liposome into the bloodstream ofan animal in a location to flow through a target site in said animal,said first fluorescent dye being releasable from said first heatsensitive liposome at a temperature of about 45° C. to 49° C., andheating said tissue in said target site to a temperature to release saidfirst fluorescent dye and fluorescing said first fluorescent dye toindicate and visualize a tissue temperature when said tissue reaches atemperature of at least 45° C., and continuing heating said tissue insaid target site for a time sufficient to hyperthermally heat treat saidtissue and kill cells in said tissue in said target site and at atemperature below a protein denaturing temperature.
 9. The method ofclaim 8, comprising heating said target site to a temperature betweenabout 47° C. and about 49° C. for about 1-10 minutes.
 10. The method ofclaim 8, wherein said first liposome encapsulates a bioactive compound,and wherein said method comprises heating said first liposome to releasesaid bioactive compound.
 11. The method of claim 10, wherein saidbioactive compound is heat activated.
 12. The method of claim 10,wherein said bioactive compound is an antiproliferative agent or anantitumor agent.
 13. The method of claim 10, wherein said bioactiveagent is selected from the group consisting of cisplatin, carboplatin,tetraplatin, iproplatin, adriamycin, mitomycin C, actinomycin,ansamitocin and bleomycin.
 14. The method of claim 8, wherein said heatsource is a laser source, a microwave source, an infrared source or anultrasonic source.
 15. The method of claim 8, wherein said heat sourceis a source of heated fluid and said method comprises applying saidheated fluid to said target site.
 16. A method of hyperthermallyheat-treating tissue and killing cells in an animal, said methodcomprising the steps of: providing a temperature indicating substanceincluding a fluorescent dye encapsulated within a heat sensitiveliposome, said fluorescent dye being releasable from said liposome at atemperature of about 45° C. to about 49° C., and introducing saidtemperature indicating substance into the bloodstream of said animal toflow through said tissue in a target site, and applying a heat source tosaid tissue at said target site and hyperthermally heating said tissuein said target site to at least 47° C. to kill cells in said tissue bythe application of heat from said heat source without denaturingproteins in said tissue, and releasing, fluorescing and visualizing saiddye to indicate that a predetermined tissue temperature has beenattained at said target site.